Fig. 3
Resected forks are able to restart in BRCA2-deficient cells and lead to increased chromosomal aberrations. a Expression of RAD51 after siRNA knockdown in U2OS cells. b Size distribution of IdU tract length in BRCA2-deficient and -proficient U2OS cells in the presence and absence of HU. Cells were transfected with control siRNA (siNEG), RAD51 siRNA, or BRCA2 siRNA before IdU and CldU labeling. Out of 2 repeats; n ≥ 250 tracts scored for each data set. Bars represent the median. Statistics: Mann–Whitney; ****P < 0.0001. c Schematic of the single-molecule DNA fiber tract analysis for the fork restart experiments. Red-green contiguous tracts (restarting forks). Red only tracts (stalled/terminated forks). d Quantification of restarting forks in BRCA2-deficient and -proficient U2OS cells with or without RAD51 siRNA knockdown. Out of 2 repeats, the percentage is established on at least 250 tracts scored for each data set. Mean shown. Errors, S.E.M. Statistics: unpaired t test; **P < 0.01. e Quantification of restarting forks in BRCA2-deficient and -proficient U2OS cells with or without EXO1 siRNA knockdown. Mirin (50 μM) was added concomitantly with HU treatment, as indicated. Out of 2 repeats, the percentage is established on at least 250 tracts scored for each data set. Mean shown. Errors, S.E.M. f Size distribution of CldU tract length in BRCA2-deficient and -proficient U2OS cells in the presence of HU. Cells were transfected with control siRNA (siNEG), EXO1 siRNA, or BRCA2 siRNA before IdU and CldU labeling. Mirin (50 μM) was added concomitantly with HU treatment, as indicated. Out of 2 repeats; n ≥ 250 tracts scored for each data set. Bars represent the median. Statistics: Mann–Whitney; ****P < 0.0001. g Chromosomal aberrations in BRCA2-deficient and -proficient U2OS cells in the presence of HU. Left, representative images of metaphase spreads in the presence of HU. Scale bar, 25 μm. Sketch above the images delineates experimental design. Right, numbers of chromosomal aberrations per metaphase are plotted. At least 50 metaphases counted in each experiments. Mean shown, n = 3.. Errors, S.E.M. Statistics: unpaired t test: ****P < 0.0001
